This invention concerns the expression of polynucleotides and, in particular, the JeT promoter.
Metabolic engineering of living organisms is still in its infancy with respect to industrial applications, despite the fact that genetic engineering is now feasible. To a large extent, this may be due to the disappointing outcome of many of the attempts so far to improve strain performance. There are at least two reasons for the negative outcome of the attempts to increase metabolic fluxes:
One is that the genetic engineer tends to overlook the subtlety of control and regulation of cellular metabolism. For example, a branching flux in a pathway can be eliminated by deleting a gene. Quite often, this will have secondary effects on the metabolism, for instance by lowering metabolite concentrations that are essential to other parts of the cellular metabolism (e.g. processes that are essential to the growth of the organism) and the net result may be that the overall performance of the cell with respect to the desired product is decreased. Instead, it is necessary to tune the expression of the relevant gene around the normal expression level and determine the optimal expression level, for instance as the level that maximizes or minimizes the flux.
The second reason for the negative outcome lies in the rate-limiting concept itself: both metabolic control theory (Kacser and Burns, 27 Symp. Soc. Exp. Biol. 65104 (1973)) and experimental determinations of control by individual steps in a pathway (Schaaff et al., 5 Yeast 285-290 (1989); Jensen et al., 12. EMBO J. 1277-1282 (1993)) have shown that reaction steps which were expected to be rate limiting with respect to a particular flux, turned out to have no or very little control over the flux. Instead, the control and regulation of the cellular metabolism turned out to be distributed over several enzymes in a pathway, and it may be necessary to enhance the expression of several enzymes in order to obtain a higher flux. According to metabolic control theory, the total flux control exerted by all the enzymes in a pathway, should always sum up to 1. Therefore, after one enzyme concentration has been optimized, the flux control will have shifted to another enzyme, and it may then be useful to perform additional rounds of enzyme optimization in order to increase the flux further.
In summary, flux optimization requires (1) fine-tuning of enzyme concentration rather than many fold over expression and often (2) optimization of the level of several enzymes in a pathway rather than looking for the rate limiting step. As a result, there is a need for promoters that can be useful for addressing flux optimization.
The JeT promoter (SEQ. ID. NO:1) takes advantage of a unique combination of transcription factor binding sites resulting in transcriptional activity comparable to a number of strong mammalian promoters such as the simian virus 40 (SV40) and ubiquitin (UbC) promoters. The promoter consists of five key elements:
(1) a TATA box (TATATAA) (SEQ. ID. NO:2),
(2) a transcription initiation site (Inr) (CTAGTTC) (SEQ. ID. NO:3),
(3) a CAT consensus sequence (CCAAT) (SEQ. ID. NO:4) in conjunction with
(4) a CArG element (CCTTTTATGG) (SEQ. ID. NO:5) and finally,
(5) four Sp1 transcription binding sites (GGGCGG) (SEQ. ID. NO:6) arranged in two tandems. The CAT/CArG complex is also referred to as a Serum Response Element (SRE). The JeT promoter sequence is depicted in FIG. 1 with the consensus boxes marked.